The goal of this project is to understand how mRNAs, proteins, and subcellular organelles are distributed and organized in muscle and nerve cells. In multinucleated muscle cells, the nonuniform distribution of specific mRNAs and proteins contributes to the formation of the neuromuscular junction. In neurons, differential distribution of specific mRNAs may play a role in the establishment and maintenance of axonal~dendritic polarity. We are trying to understand how mRNA stability and mRNA translation influence mRNA localization. To that effect, we are localizing, by in situ hybridization, both endogenous and foreign mRNAs from transfected genes, in the mouse muscle cell line C2, and in primary cultures of rat hippocampal neurons. In the past year, we have focused our work on the endogenous transferrin receptor (TfR). It was found that at both mRNA and protein levels, distribution of TfR mRNA is not uniform in myotubes. Localization of TfR mRNA appeared independent of iron concentration in the medium, a modulator of TfR mRNA stability, indicating that distribution of TfR mRNA is independent of its half~life. On the other hand, treatment with protein synthesis inhibitors suggested that attachment to ribosomes on the rough endoplasmic reticulum is, at least in part, responsible for localization of TfR mRNA. We will now try to identify sequences responsible for this localization by transfection of chimeric TfR cDNAs. We have shown that TfR mRNA in neurons is confined to the cell body and will now determine if translation is part of this localization. An important advance with the neuron work has been the development of a modified protocol for transfection of neurons with the lipid reagent DOTAP, resulting in a five~fold increase in transfection efficiency. We are continuing to investigate the mechanism of vesicle and protein traffic in muscle by studying the localization of the glucose transporter GLUT4 and its translocation to the plasma membrane following stimulation by insulin or exercise. An important result was to find, contrary to previous reports, that C2 cells express GLUT4 as part of their normal differentiation program. Young myotubes appear unable to translocate GLUT4 to the cell surface, but mature myotubes undergoing spontaneous contraction, appear capable of doing so. We are presently investigating the mechanism that regulates GLUT4 translocation in C2 cells.